This application relates to purified laminin 8 and methods for its use.
Basal laminae (basement membranes) are sheet-like, cell-associated extracellular matrices that play a central role in cell growth, tissue development, and tissue maintenance. They are present in virtually all tissues, and appear in the earliest stages of embryonic development.
Basal laminae are central to a variety of architectural and cell-interactive functions: (See for example, Malinda and Kleinman, Int. J. Biochem. Cell Biol. 28:957-959 (1996); Aumailley and Krieg, J. Invest. Dermatology 106:209-214 (1996)).
1. They serve as architectural supports for tissues, providing adhesive substrata for cells.
2. They create perm-selective barriers between tissue compartments that impede the migration of cells and passively regulate the exchange of macromolecules. These properties are illustrated by the kidney glomerular basement membrane, which functions as an important filtration structure, creating an effective blood-tissue barrier that is not permeable to most proteins and cells.
3. Basal laminae create highly interactive surfaces that can promote cell migration and cell elongation during embryogenesis and wound repair. Following an injury, they provide a surface upon which cells regenerate to restore normal tissue function.
4. Basal laminae present information encoded in their structure to contacting cells that is important for differentiation and tissue maintenance. This information is communicated to the cells through various receptors that include the integrins, dystroglycan, and cell surface proteoglycans. Signaling is dependent not only on the presence of matrix ligands and corresponding receptors that interact with sufficient affinities, but also on such topographical factors as ligand density in a three-dimensional matrix xe2x80x9clandscapexe2x80x9d, and on the ability of basal lamina components to cluster receptors. Because these matrix proteins can be long-lived, basal laminae create a xe2x80x9csurface memoryxe2x80x9d in the basal lamina for resident and transient cells.
The basal lamina is largely composed of laminin and type IV collagen heterotrimers that in turn become organized into complex polymeric structures. To date, six type IV collagen chains and at least twelve laminin subunits have been identified. These chains possess shared and unique functions and are expressed with specific temporal (developmental) and spatial (tissue-site specific) patterns.
Laminins are a family of heterotrimeric glycoproteins that reside primarily in the basal lamina. They function via binding interactions with neighboring cell receptors, and by forming laminin networks, and they are important signaling molecules that can strongly influence cellular function. Laminins are important in both maintaining cell/tissue phenotype as well as promoting cell growth and differentiation in tissue repair and development.
Laminins are large, multi-domain proteins, with a common structural organization. The laminin molecule integrates various matrix and cell interactive functions into one molecule.
The laminin molecule is comprised of an xcex1-, xcex2-, and xcex3-chain subunit joined together through a coiled-coil domain. Within this structure are identifiable domains that possess binding activity towards other laminin and basal lamina molecules, and membrane-bound receptors. Domains VI, IVb, and IVa form globular structures, and domains V, IIIb, and IIIa (which contain cysteine-rich EGF-like elements) form rod-like structures. (Kamiguchi et al., Ann. Rev. Neurosci. 21:97-125 (1998)) Domains I and II of the three chains participate in the formation of a triple-stranded coiled-coil structure (the long arm).
Table 1 shows the individual chains that each laminin type is composed of:
Four structurally-defined family groups of laminins have been identified. The first group of five identified laminin molecules all share the xcex21 and xcex31 chains, and vary by their xcex1-chain composition (xcex11 to xcex15 chain). The second group of five identified laminin molecules all share the xcex22 and xcex31 chain, and again vary by their xcex1-chain composition. The third group of identified laminin molecules has one identified member, laminin 5, with a chain composition of xcex13xcex23xcex32. The fourth group of identified laminin molecules has one identified member, laminin 12, with the newly identified xcex33 chain (xcex12xcex21xcex33).
Some progress has been made in elucidating the relationship between domain structure and function. (See, for example, Wewer and Engvall, Neuromusc. Disord. 6:409-418 (1996).) The overall sequence similarity among the homologous domains in different chains varies, but it is highest in domain VI (thought to play a key role in laminin polymerization), followed by domains V (possibly involved in protein-protein interactions) and III (entactin/nidogen binding; possible cell adhesion sites), and is lowest in domains I, II (both thought to be involved in intermolecular assembly, and containing possible cell adhesion sites), and G. Not all domains are present in all 3 types of chains. The globular G domain (thought to be involved in cell receptor binding) is present only in the xcex1 chains. Other domains may not be present in all chains within a certain chain type. For example, domain VI is absent from xcex13, xcex14, and xcex32 chains. (Wewer and Engvall, 1996)
As a result of their large size ( greater than 600 kD) and unique structure, the laminin molecules can be resolved in the electron microscope. (Wewer and Engvall, 1996) Typically, laminins appear as cross-shaped molecules in an EM. The three short arms of the cross represent the amino terminal portions of each of the three separate laminin chains (one short arm per chain). The long arm of the cross is composed of the C-terminal parts of the three chains, which together form a coiled coil structure. (Wewer and Engvall, 1996) The long arm ends with the globular G domain.
The coiled-coil domain of the long arm is crucial for assembly of the three chains of laminin. (Yurchenco et al., Proc. Natl. Acad. Sci. 94:10189-10194 (1997)). Disulfide bonds bridge and stabilize all three chains in the most proximal region of the long arm and join the xcex2 and xcex3 chains in the most distal region of the long arm.
A model of laminin receptor-facilitated self-assembly, based on studies conducted with cultured skeletal myotubes and Schwann cells, predicts that laminins bind to their receptors, which freely diffuse in a fluidic membrane, when ligand-free. Receptor engagement forces the laminins into a high local two-dimensional concentration, facilitating their mass-action driven assembly into ordered surface polymers. In this process, the engaged receptors are also reorganized, accompanied by cytoskeletal rearrangements. (Colognato, J. Cell Biol. 145:619-631 (1999)) This reorganization activates the receptors, causing signal transduction with the alteration of cell expression, shape and/or behavior. The evidence is that laminins must possess both cell-interacting and architecture-forming sites, which are located in different protein domains and on different subunits.
One class of laminin receptors are the integrins, which are cell surface receptors that mediate many cell-matrix and cell-cell interactions. Integrins are heterodimers, consisting of an xcex1 and a xcex2 subunit. 16 xcex1- and 8 xcex2-subunits are known, and at least 22 combinations of xcex1 and xcex2 subunits have been identified to date. Some integrins have only one or a few known ligands, whereas others appear to be very promiscuous. Binding to integrins is generally of low affinity, and is dependent on divalent cations. Integrins, activated through binding to their ligands, transduce signals via kinase activation cascades, such as focal adhesion and mitogen-activated kinases. Several different integrins bind different laminin isoforms more or less specifically. (Aumailley et al., In The Laminins, Timpl and Ekblom, eds., Harwood Academic Publishers, Amsterdam. pp. 127-158 (1996))
Laminin 8, a recently identified laminin, is composed of xcex14, xcex21, and xcex31 laminin chains. The laminin xcex14 chain is widely distributed both in adults and during development. (Iivanainen et al., J. Biol. Chem. 272:27862-27868 (1997)) In adults it is found in the basement membrane surrounding cardiac, skeletal, and smooth muscle fibers, and in lung alveolar septa. Furthermore, it is found in the endothelial basement membrane both in capillaries and larger vessels, and in the perineurial basement membrane of peripheral nerves, as well as in intersinusoidal spaces, large arteries, and smaller arterioles of bone marrow. (Frieser et al., Eur. J. Biochemistry 246:727-735 (1997); Miner et al., J. Cell Biol. 137:685-701 (1997); Geberhiwot et al., Exptl. Cell Res. 253:723-732 (1999); Gu et al., Blood 93:2533-2542 (1999); Iivanainen et al., J. Biol. Chem. 272:27862-27868 (1997)).
Laminin 8 is a major laminin isoform in the vascular endothelium (Iivanainen et al., J. Biol. Chem. 272:27862-27868 (1997); Frieser et al., 1997), is expressed and adhered to by platelets (Geberhiwot et al., Exptl. Cell Res. 253:723-732 (1999)), and is the only laminin isoform synthesized in 3T3-L1 adipocytes, with its level of synthesis shown to increase upon adipose conversion of the cells. (Niimi et al., Matrix Biology 16:223-230 (1997)) Laminin 8 was further speculated to be the laminin isoform generally expressed in mesenchymal cell lineages to induce microvessels in connective tissues. (Niimi et al., 1997).
Laminin 8 has also been identified in mouse bone marrow primary cell cultures, arteriolar walls, and intersinusoidal spaces where data indicated that it is the major laminin isoform in the developing bone marrow. (Gu et al., Blood 93:2533-2542 (1999). The investigators concluded that, due to its localization in adult bone marrow adjacent to hematopoietic cells, laminin isoforms containing the xcex14 chain are the most likely to have biologically relevant interactions with developing hematopoietic cells. (Gu et al., 1999)
Despite the broad tissue distribution of the laminin xcex14 chain and laminin 8, there is not a means to prepare substantially purified laminin 8 from cell or tissue sources for research and therapeutic purposes, nor has a means for recombinant expression of laminin 8 been developed. Such research and therapeutic purposes include, but are not limited to, methods for treating-injuries to tissue of mesenchymal origin, such as bone, cartilage, tendon, and ligament, treating injuries to vascular tissue, promoting cell attachment and migration, promoting therapeutic angiogenesis and neural regeneration, ex vivo cell therapy, improving the biocompatibility of medical devices, and preparing improved cell culture devices and media.
Thus, there is a need in the art for adequate amounts of substantially purified laminin-8 for research and therapeutic purposes, and methods for making laminin 8. Such laminin 8 could be prepared either from cell lines in culture, or via recombinant DNA technology.
A preferred method of production is the use of recombinant DNA technology to engineer a human cell line of choice to produce recombinant laminin-8 (xe2x80x9cr-laminin 8xe2x80x9d). A recombinant-based method of laminin-8 production has several advantages over purification from human tissue or isolation from human cell lines in culture:
1. The recombinant produced protein is free of human pathogens. While this is also true for endogenous cell culture produced protein, protein derived from human tissue carries a risk for contamination by HIV, hepatitis, and other infectious agents.
2. Expression levels of the protein, and hence yields, can be improved through the use of genetically engineered genes/vectors that enhance the production of the encoded protein.
3. It is possible to engineer additional peptide sequences to the protein chain that provides a binding site for a commercially viable affinity purification procedure.
4. The method can provide for the modification of protein structure/function through the addition, substitution, elimination, and/or other modifications of protein domain structures. For example, it may be desirable to introduce an integrin binding site (e.g. RGD), switch integrin recognition sites, or engineer in a stable binding site to a synthetic substrate. Thus, the creation of expression vectors that express laminin chains generates enormous flexibility for future uses and creates a basis for creating second generation xe2x80x9cdesignerxe2x80x9d laminins.
The present invention fulfills the need in the art for a source of substantially purified laminin 8 protein, methods for making substantially purified recombinant laminin 8 (hereinafter referred to as r-laminin 8), pharmaceutical compositions comprising laminin 8, and methods of using laminin 8 for treating injuries to tissue of mesenchymal origin, such as bone, cartilage, tendon, and ligament, treating injuries to vascular tissue, promoting cell attachment and migration, ex vivo cell therapy, improving the biocompatibility of medical devices, and preparing improved cell culture devices and media.
In one aspect, the present invention provides recombinant host cells that express laminin 8 chains and secrete r-laminin 8. In another aspect, the present invention provides substantially purified laminin 8, and methods for producing substantially purified r-laminin 8.
In a further aspect, the present invention provides pharmaceutical compositions, comprising laminin 8 together with a pharmaceutically acceptable carrier. Such pharmaceutical compositions can optionally be provided with other extracellular matrix components.
In further aspect, the present invention provides methods and kits for accelerating the healing of injuries to tissue of mesenchymal origin, such as bone, cartilage, tendon, and ligament, treating injuries to vascular tissue, and for improving the biocompatibility of grafts used for treating such injuries. In specific examples, laminin 8 or pharmaceutical compositions thereof are used to:
a. promote re-endothelialization at the site of vascular injuries;
b. improve the xe2x80x9ctakexe2x80x9d of grafts;
c. improve the biocompatibility of medical devices;
d. treat neural injuries (neural regeneration);
e. regulate angiogenesis; and
d. promote cell attachment and migration
by providing an amount effective of r-laminin 8 for the various methods. In preferred embodiments of all of these methods, recombinant laminin 8 is used. The kits comprise an amount of laminin 8 effective for the desired effect, and instructions for the use thereof.
In a further aspect, the present invention provides improved medical devices and grafts, and methods for preparing improved medical devices and grafts, wherein the improvement comprises applying an amount effective of laminin 8 or the pharmaceutical compositions of the invention to the device or graft for the desired application.
In a further aspect, the invention provides improved cell culture devices, and methods for preparing improved cell culture devices, for the growth and maintenance of cells in culture, by providing an amount effective of laminin 8 for the attachment of cells to a cell culture device for the subsequent proliferation/differentiation/stasis of the cells.
In another aspect, the invention provides a cell culture growth supplement, comprising laminin 8. In another aspect, the invention provides an improved cell culture growth media, wherein the improvement comprises the addition of r-laminin 8.